materials and methods : non-diabetic twenty-seven cad patients (mean ± se age: 60.6± 8.99)...

MATERIALS AND METHODS : Non-diabetic twenty-seven CAD patients (mean ± SE age: 60.6± 8.99) undergoing elective cardiac surgery and twenty-three without CAD patients (controls) (mean ± SE age: 55.1± 6.9) undergoing valve replacement were prospectively enrolled in this study. Study groups were divided into two subgroups; 1. body mass index (BMI) of 27kg/cm2 or less and 2. BMI greater than 27 kg/cm2. The cutoff value of 27 kg/cm2 corresponds to a threshold for abnormal glucose and increased mortality. Paired sample biopsies of EAT, PAT and SAT were obtained during the surgery. EAT samples were collected from anterior surface of right ventricule while PAT samples were from were from the fat within the mediastinum, outside the pericardial sac and SAT were obtained from retrosternal region. Following the homogenisation, mRNA expression levels of 11–HSD-1, Glucocorticoid receptor (GCR), plasminogen activator inhibitor-1 (PAI-1), angiotensinogen (ANG), C3, angiotensin receptor-2 (ANGR2), leptin (LEP), leptin receptor (LEPR), vascular endothelial growth factor (VEGF), adiponectin (ADPQ), retinol binding protein-4 (RBP-4), CD68, CD45, six-transmembrane protein of prostate 2 (STAMP2), tumor necrosis factor- (TNF-), fatty acid binding protein-4 (FABP-4) were determined by SYBR green-based qRT-PCR using a LightCycler 480 (Roche-Germany) instrument. Fasting venous blood samples were separated as serum, plasma and cellular portions. Serum hs-CRP, TNF-α, IL-6 IL-18 and leptin levels were assayed by ELISA.

Aim: In the light of the previous studies indicating the involvement of some epicardial adipokines in the development of obesity and CAD, we proposed to determine whether there was any difference between the three different fat depots; epicardial, mediastinal or paracardial and subcutaneous adipose tissues (EAT, PAT and SAT, respectively) in non-diabetic patients with CAD and non-CAD in terms of sixteen related adipokines messenger RNA and their associations with serum levels of inflammatory markers.

CARDIAC VISCERAL AND SUBCUTANEOUS ADIPOSE TISSUE ADIPOKINES ARE DYSREGULATED IN CARDIAC VISCERAL AND SUBCUTANEOUS ADIPOSE TISSUE ADIPOKINES ARE DYSREGULATED IN OBESITY AND CORONARY ARTERY DISEASEOBESITY AND CORONARY ARTERY DISEASE

Fatmahan Atalar1, Selcuk Gormez2, Barıs Caynak3, Gökçe Akan4, Belhhan Akpinar3, Demet Gunay5

A.Sevim Buyukdevrim6 and Zeliha Yazıcı7

1Istanbul University, Istanbul Medical Faculty, Child Health Institute, Growth-Development and Pediatric Endocrinology Department, Istanbul, Turkey, 2Acibadem Hospital, Cardiology Dept, Istanbul, Turkey, 3Istanbul Bilim University, Faculty of Medicine, Dept. of Cardiovascular Surgery and Florence Nightingale Hospital, Istanbul, Turkey, 4Istanbul Bilim University, Medical Biology and Genetics Dept, Istanbul, Turkey, 5 Florence Nightingale Hospital, Biochemistry Laboratory, Istanbul, Turkey, 6Emeritus, Istanbul University, Istanbul Medical School, Dept. of Internal Medicine, Section of Diabetes, Istanbul, Turkey, 7Istanbul University, Cerrahpasa Medical School, Pharmacalogy Dept. Istanbul, Turkey,

BACKGROUND: Coronary heart disease is one of the most common type of heart disease and the leading cause of mortality worldwide. A close association between obesity and increased cardiovascular diseases has been established by the epidemiological data, even though the molecular pathways underlying this close association still remain unclear. Cytokines (e.g. tumor necrosis factor-α (TNF- α), interleukin (IL)-6, plasminogen activator inhibitor (PAI)-1 ), chemokines (e.g. IL-8) and hormones (e.g leptin, adiponectin, resistin) secreted by the adipose tissues are the members of the adipokine family and they all mediate this association by regulating lipid accumulation, inflammation and atherogenesis. Pathogenic gene expression profile of adipokines have been demonstrated in epicardial adipose tissue (EAT), omental adipose tissue (OAT) and subcutaneous adipose tissue (SAT) of CAD patients indicating the contribution of the local and systemic chronic infammation to the pathogenesis of CAD (1,2).

Table 1. Patient Characteristics

BMI indicates body mass index ; CAD, coronary artery disease; ACEI/ARB, angiotensin converting enzyme inhibitor/angiotensin receptor blocker; CCB, calcium channel blocker;HOMA-IR, Homeostasis Model Assessment-Insulin Resistance; hs-CRP, high-sensitivity C-Reactive Protein.§ P values represent comparisons using a 1-way ANOVA or Kruskal- Wallis, χ2, or Fisher exact test as appropriate.‡ The data are expressed as percentages of patients.The data are expressed as mean values plus or minus the standard deviatiation unless otherwise stated.

0

2

4

6

8

10

12

14

16

18

Control<27kg/m2 Control>27kg/m2 CAD<27kg/m2 CAD>27kg/m2

Lev

els

of e

xpre

ssio

n of

mR

NA

(A

rbit

rary

Uni

ts)

ADIPOQ(EAT)ADIPOQ(PAT)ADIPOQ(SAT)

0

0,5

1

1,5

2

2,5

3


Lev

els

of

exp

ress

ion

of

mR

NA

(A

rbitr

ary

Un

its)

AGTR2(EAT)AGTR2(PAT)AGTR2(SAT)

0

0,5

1

1,5

2

2,5

3


Lev

els

of e

xpre

ssio

n of

mR

NA

(A

rbit

rary

Uni

ts)

AGT(EAT)AGT(PAT)AGT(SAT)

0

10

20

30

40

50

60

70


Lev

els

of e

xpre

ssio

n of

mR

NA

(A

rbit

rary

Uni

ts)

FABP4(EAT)FABP4(PAT)FABP4(SAT)

0

0,2

0,4

0,6

0,8

1


Lev

els

of e

xpre

ssio

n of

mR

NA

(A

rbit

rary

Uni

ts)

CD45(EAT)CD45(PAT)CD45(SAT)

0

0,4

0,8

1,2

1,6

2


Lev

els

of

exp

ress

ion

of

mR

NA

(A

rbit

rary

Un

its)

C3(EAT)C3(PAT)C3(SAT)

0

0,2

0,4

0,6

0,8

1


Lev

els

of e

xpre

ssio

n of

mR

NA

(A

rbit

rary

Uni

ts)

CD68(EAT)CD68(PAT)CD68(SAT)

0

0,4

0,8

1,2

1,6

2


Lev

els

of

exp

ress

ion

of

mR

NA

(A

rbit

rary

Un

its)

LEP(EAT)LEP(PAT)LEP(SAT)

0

1

2

3

4

5

6


Leve

ls o

f exp

ress

ion

of m

RN

A (A

rbitr

ary

Uni

ts)

GCR(EAT)GCR(PAT)GCR(SAT)

0

2

4

6

8

10

12

14

16

18

20


Lev

els

of

exp

ress

ion

o

f m

RN

A (

Arb

itra

ry U

nit

s) PAI-1(EAT)PAI-1(PAT)PAI-1(SAT)

0

2

4

6

8

10

12

14

16

18

20


Lev

els

of

exp

ress

ion

o

f m

RN

A (

Arb

itra

ry U

nit

s)

RBP4(EAT)RBP4(PAT)RBP4(SAT)

0

1

2

3

4

5


Lev

els

of e

xpre

ssio

ns o

f m

RN

A (

Arb

itra

ry U

nits

)

STAMP2(EAT)STAMP2(PAT)STAMP2(SAT)

0

0,2

0,4

0,6

0,8

1


Lev

els

of e

xpre

ssio

ns o

f m

RN

A (

Arb

itra

ry U

nits

)

VEGF(EAT)VEGF(PAT)VEGF(SAT)

0

0,2

0,4

0,6

0,8

1


Lev

els

of e

xpre

ssio

ns o

f m

RN

A (

Arb

itra

ry U

nits

)

TNF(EAT)TNF(PAT)TNF(SAT)

0

0,1

0,2

0,3

0,4

0,5


Lev

els

of e

xpre

ssio

n of

mR

NA

(A

rbit

rary

Uni

ts) LEPR(EAT)

LEPR(PAT)LEPR(SAT)

0

1

2

3

4

5


Lev

els

of

exp

ress

ion

of

mR

NA

(A

rbit

rary

Un

its)

11β HSD1(EAT)11β HSD1(PAT)11β HSD1(SAT)

Figure 1: mRNA levels of 11–HSD-1, GCR, PAI-1, ANG, C3,ANGR2, LEP, LEPR,VEGF, ADPQ, RBP-4, CD68, CD45, STAMP2, TNF- and FABP-4 in controls with BMI≤27kg/m-2, controls with BMI>27kg/m-2, CAD with BMI ≤ 27kg/m-2 and CAD with BMI>27kg/m-2

Figure 2:A. Circulating levels of hs-CRP, B.TNF-alpha, C.leptin, D. IL-6, E.adiponectin and F.IL-18 in controls with BMI≤27kg/m-2, controls with BMI>27kg/m-2, CAD with BMI ≤ 27kg/m-2 and CAD with BMI>27kg/m-2. *p ≤ 0.05,**p≤0.01

0

0,5

1

1,5

2

2,5

3

Controls

BMI≤27

Controls

BMI≥27

CAD

BMI≤27

CAD

BMI≥27

Seru

m levels

of H

s-C

RP

(m

g/

dL

)

Hs-CRP(mg/ dL)

0

1

2

3

4

5

6

7

8

9

Controls

BMI≤27

Controls

BMI≥27

CAD

BMI≤27

CAD BMI≥27

Seru

m levels

of I

L-6 (

pg/

mL

)

IL-6 (pg/ mL)

0

50

100

150

200

250

300

350

400

450

Controls

BMI≤27

Controls

BMI≥27

CAD

BMI≤27

CAD

BMI≥27

Seru

m levels

of I

L-18(p

g/

mL

)

IL-18(pg/ mL)

0

0,5

1

1,5

2

2,5

3

Controls

BMI≤27

Controls

BMI≥27

CAD

BMI≤27

CAD

BMI≥27

Seru

m levels

of T

NF-alp

ha (

pg/m

L)

TNF-alpha (pg/ mL)

0

1

2

3

4

5

6

7

8

9

10

Controls

BMI≤27

Controls

BMI≥27

CAD

BMI≤27

CAD

BMI≥27

Seru

m levels

of L

ep

tin

(ng/

mL

)

Leptin (ng/ mL)

0

2

4

6

8

10

12

14

Controls

BMI≤27

Controls

BMI≥27

CAD

BMI≤27

CAD

BMI≥27

Seru

m levels

of A

dip

onectin

(u

g/

mL

)

Adiponectin (ug/ mL)

A B

FE

DC** **

**

****

**

** *

RESULTS

Conclusions:• We confirm that EAT, PAT and SAT are abundant sources of adipokines. •IL-18 levels are positively associated with obesity and CAD, though adiponectin levels are negatively associated. Leptin levels are associated with CAD alone and obesity related CAD, while TNF-α levels with obesity.•CAD and obesity are differentially associated with the expressions of EAT, PAT and SAT adipokines. •The epicardial, mediastinal and subcutaneous expressions of 11β-HSD-1, AGT, CD68, FABP4, GCR, LEP and RBP4 were found to be associated with CAD and obesity.

•EAT expression of ADIPOQ, AGTR2,C3,PAI-1 and CD45 were related with obesity while TNF-α, STAMP2 and VEGF expressions with CAD. •PAT expression of ADIPOQ, AGTR2 and CD45 were related to obesity while PAI-1 and STAMP2 expressions with CAD.•SAT expression of AGTR2, C3, CD45 and PAI-1 were related with obesity while CD68, STAMP2, TNF- α and VEGF expressions with CAD.

•The study is still ongoing to determine new cross-talks between pathways and the disease specific pathway regulators in order to explain the tissue specific expression of the adipokines which eventually lead to different diseases.

Control Group CAD Group

Characteristics BMI≤27 kg/m2 BMI>27 kg/m2

BMI≤27kg/m2 BMI>27 kg/m2 P value§

N

Age, years

10

54.2±7.9

17

56.3±5.7

13

59.7±9.2

12

60.7±9.0 NS

BMI 25.3±2.7 33.9±3.6 23.8±0.9 32.3±3.0 <0.001

Waist circumference ,cm 88.5±10.7 100.8±18.3 96.5±11.1 107.8±8.9 <0.001

Systolic,mm Hg 124.1±10.2 125±13.1 132.5±10.4 131.9±17.3 NS

Diastolic,mm Hg 75.5±6.9 76.9±9.6 75.0±12.9 79.0±8.4 NS

Total Cholesterol,mg/dL 202±40.0 195.5±61.3 199.5±15.1 175.5±40.0 NS

HDL 44.8±8.7 45.1±12.7 40.3±5.4 41.9±9.9 NS

LDL 133.3±27.4 126.5±52.3 122.5±17.0 107.9±37.5 NS

Triglycerides,mg/dL 142.3±73.9 134.9±58.1 223.5±80.3 152.3±68.8 NS

Fasting glucose,mg/mL 77±16 101±25 91±34 106±21 0.02

HOMA-IR 1.1±0.5 2.8±0.7 1.8±1.3 2.5±1.6 0.01

Dyslipidemia‡ 33.0 62.0 71.0 89.0 <0.001

Smoking status ex/current‡ 20.0/60.0 30.0/65.0 46.0/15.0 83.0/33.0 NS

Family history of CAD‡ 11.0 24.0 54.0 72.0 NS

Medication

Aspirin‡ 62.0 55.0 88.0 79.0 <0.001

Statin‡ 46.0 34.0 61.0 82.0 <0.001

ACEI/ARB‡ 22.0 48.0 36.0 46.0 NS

Calcium Channel Blocker 16.0 13.0 19.0 50.0 <0.001

β-Blocker‡ 46.0 60.0 73.0 80.0 NS

Fat (cm3)

Epicardial fat 4.1±2.2 4.5±2.5 3.8±1.1 8.3±4.5 0.002

Omental fat 71.3±37.0 94.5±29.7 63.0±8.9 109.5±42.7 0.02

Subcutaneous fat 109.8±50.9 157.8±68.8 80.7±20.2 149.8±58.1 0.05

Abdominal fat 184.9±77.8 255.7±88.8 143.6±18.9 256.6±67.1 0.006

Serum levels of inflammatory markers and adipokines

Hs-CRP,mg/dL 1.3±1.1 2.5±0.6 1.1±0.4 1.9±0.7 NS

TNF-alpha, pg/mL 1.1±0.5 1.9±0.62 1.9±0.5 2.3±1.2 0.04

IL-6, pg/mL 3.5±1.3 4.4±0.6 5.1±2.9 7.2±1.4 0.01

IL-18, pg/mL 158.3±29.6 199.4±41.1 160.6±24.4 354.1±50.6 0.005

Leptin, ng/mL 4,3±0.2 5,7±0.4 7,1±0.3 8,2±0.4 0.01

Adiponectin, μg/mL 9,21±1.6 10,68±1.0 8,57±3.2 6,25±2.4 0.01

0552

This work is supported by Turkish Diabetes Foundation and Istanbul University BAP.

References1. F. Atalar, S. Gormez, B. Caynak, G. Akan, G. Tanriverdi, S. Bilgic-Gazioglu, D. Gunay, C. Duran, B. Akpinar, U. Ozbek, A.S. Buyukdevrim, Z. Yazici. “The role of mediastinal adipose tissue 11β-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor expressions in the

development of coronary atherosclerosis in obese patients with coronary artery disease; correlation with fatty acid content-a case control study”, Cardiovas Diabetol, 11:115 (2012).2. F. Atalar, S. Gormez, B. Caynak, G. Akan, G. Tanriverdi, S. Bilgic-Gazioglu, D. Gunay, C. Duran, B. Akpinar, U. Ozbek, A.S. Buyukdevrim, Z. Yazici. “Mediastinal adipose tissue expresses a pathogenic profile of 11 β- hydroxysteroid dehydrogenase type 1, glucocorticoid receptor

and CD68 in patients with coronary artery disease”, Cardiovasc Pathol, 21(5): 365-375 (2013).

materials and methods : non-diabetic twenty-seven cad patients (mean ± se age: 60.6± 8.99)...

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